During terminal erythropoiesis, erythroid cells produce significant quantities of heme and heme intermediates which must be coupled to hemoglobin production and iron uptake. Dysregulation of heme synthesis can cause toxic accumulation of heme intermediates and heme deficiency, leading to diseases such as iron overload, anemia and porphyria. We have demonstrated that mitochondrial CLPX, a member of the ubiquitious AAA+ (ATPases associated with various cellular activities) protein unfoldases family, plays a key role in erythroid differentiation by direct regulation of heme synthesis. ClpX functions as a ring-shaped homo-hexamer and is best understood for its function in a proteasome-like enzyme complex with the peptidase ClpP (the ClpXP ATP-dependent protease). CLPX regulates the heme synthesis pathway by mediating activation and degradation of the heme biosynthetic enzymes ALAS1 and ALAS2, which catalyze the committed step of heme synthesis. Beyond regulation of the ALAS enzymes, our data indicate that CLPX regulates the terminal steps of porphyrin synthesis. This finding is conceptually significant as ALA synthesis has until now been understood to be the regulated step of porphyrin synthesis. The goal of this proposal is to identify the mechanisms by which CLPX regulates erythoid heme synthesis and erythropoiesis. This will be accomplished by Specific Aim 1, which will interrogate the regulation of terminal heme synthesis enzymes by CLPX, and Specific Aim 2, which will examine the effects of CLPX deficiency on erythropoiesis within the context of mouse and zebrafish models.